Biosynthesis of coral settlement cue tetrabromopyrrole in marine bacteria by a uniquely adapted brominase–thioesterase enzyme pair

Abstract

Halogenated pyrroles (halopyrroles) are common chemical moieties found in bioactive bacterial natural products. The halopyrrole moieties of mono-and dihalopyrrole-containing compounds arise from a conserved mechanism in which a proline-derived pyrrolyl group bound to a carrier protein is first halogenated and then elaborated by peptidic or polyketide extensions. This paradigm is broken during the marine pseudoalteromonad bacterial biosynthesis of the coral larval settlement cue tetrabromopyrrole (1), which arises from the substitution of the proline-derived carboxylate by a bromine atom. To understand the molecular basis for decarboxylative bromination in the biosynthesis of 1, we sequenced two Pseudoalteromonas genomes and identified a conserved four-gene locus encoding the enzymes involved in its complete biosynthesis. Through total in vitro reconstitution of the biosynthesis of 1 using purified enzymes and biochemical interrogation of individual biochemical steps, we show that all four bromine atoms in 1 are installed by the action of a single flavin-dependent halogenase: Bmp2. Tetrabromination of the pyrrole induces a thioesterase-mediated offloading reaction from the carrier protein and activates the biosynthetic intermediate for decarboxylation. Insights into the tetrabrominating activity of Bmp2 were obtained from the high-resolution crystal structure of the halogenase contrasted against structurally homologous halogenase Mpy16 that forms only a dihalogenated pyrrole in marinopyrrole biosynthesis. Structure-guided mutagenesis of the proposed substrate-binding pocket of Bmp2 led to a reduction in the degree of halogenation catalyzed. Our study provides a biogenetic basis for the biosynthesis of 1 and sets a firm foundation for querying the biosynthetic potential for the production of 1 in marine (meta)genomes.natural products | biosynthesis | halogenation | enzymology

Organohalogens Naturally Biosynthesized in Marine Environments and Produced as Disinfection Byproducts Alter Sarco/Endoplasmic Reticulum Ca2+ Dynamics

“…23–27 Recently, the biosynthetic pathways for a number of brominated pyrroles, bipyrroles and phenols have been also elucidated. 1,19,21,28 Tetrabromopyrrole ( 31 ) isolated from Pseudoalteromonas bacteria has been shown to elicit specific development cues to coral larvae necessary for metamorphosis and benthic attachment of Orbicella franksi and Acropora palmata onto crustose coralline algae. 29,30 Remarkably, high levels of several halopyrroles, including tetrabromopyrrole ( 31 ), are produced as disinfection byproducts (DBPs) during treatment of drinking water, agricultural irrigation water and saline wastewater, and are considered as emerging pollutants because of their cytotoxicity, genotoxicity, carcinogenicity and developmental toxicity.…”

Enzymatic Halogenation and Dehalogenation Reactions: Pervasive and Mechanistically Diverse

“…While numerous crystal structures of acyl- S -CP utilizing FDHs have been reported (PltA, 112 Mpy16, 38 Bmp2, 38 CndH 108 ), key mechanistic questions remain unresolved. Primary among these is the catalytic base that abstracts the proton to rearomatize the product during the electrophilic aromatic substitution by the halenium.…”

“…Recently, the remarkable tetrahalogenating activity of the marine bacterial FDH Bmp2 was reported. 38 Bmp2 brominates all four carbon atoms of the CP-tethered pyrrole ring setting up a thioesterase mediated hydrolytic release of a 2,3,4,5-tetrabromo-pyrrole- 2 -carboxylic acid that undergoes a non-catalytic decarboxylation to yield the coral settlement chemical cue 24 (Figure 8A). 115 Multiple halogenations, thought of as relaxation in the regiospecificity for halogenation by acyl- S -CP FDHs, is in stark contrast to small molecule halogenases such as PrnA, 36 PyrH, 116 RebH, 58 and SttH 67 that display remarkable regiocontrol in halogenating tryptophan.…”

“…How an NHFe halogenase tailors this activity is not known, but the discrimination is likely encoded within the halogenase active site itself as demonstrated for FDHs. 38 …”

Enzymatic Reductive Dehalogenation Controls the Biosynthesis of Marine Bacterial Pyrroles

“…(5, 6) All Bmp biosynthetic enzymes have been characterized with the exception of an orphan ∼21 kDa protein with distant homology to thioredoxin/alkyl hydroperoxidase AhpD encoded by the open reading frame (ORF) bmp8, located upstream of CYP450 electron transport partner-encoding bmp9–10 (Figure 1A). (5, 6) Notably, bmp8 homologues are absent in the bmp gene cluster recently identified from Pseudoalteromonas sp. PS5 that produces the coral larval settlement cue 3, and not 2, as its dominant polybrominated pyrrole metabolite (Figure 1A).…”